An Aggregation-Prone Intermediate Species Is Present in the Unfolding Pathway of the Monomeric Portal Protein of Bacteriophage P22: Implications for Portal Assembly
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- 作者：Carolina A. C. A. Braga ; Danielle Carvalho ; Flá ; vio Alves Lara ; Juliana Reis Cortines ; Sean D. Moore ; Peter E. Prevelige ; Jr. ; Debora Foguel
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：June 26, 2007
- 年：2007
- 卷：46
- 期：25
- 页码：7353 - 7364
- 全文大小：481K
- 年卷期：v.46,no.25(June 26, 2007)
- ISSN：1520-4995
文摘
The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex thatserves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processesof the portal protein of P22 were investigated at different temperatures (1, 25, and 37 
C) through the useof urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterizedan intermediate species, IU, which forms at 25 C during unfolding or refolding of the portal protein in2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On theother hand, at 1 C, a two-state process is observed (
Gf = -2.2 kcal/mol). When subjected to HHP at25 or 37 C, the portal monomer undergoes partial denaturation, also forming an intermediate species,which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structureas seen by size-exclusion chromatography and electron microscopy. Again, at 1 C the unfolding inducedby HHP proved to be reversible, with Gf = -2.4 kcal/mol and V = 72 mL/mol. Interestingly, at 25C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completelyblocks its aggregation under HHP. These data are relevant to the process by which the portal proteinassembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee theproper ring formation.
C) through the useof urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterizedan intermediate species, IU, which forms at 25 C during unfolding or refolding of the portal protein in2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On theother hand, at 1 C, a two-state process is observed (Gf = -2.2 kcal/mol). When subjected to HHP at25 or 37 C, the portal monomer undergoes partial denaturation, also forming an intermediate species,which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structureas seen by size-exclusion chromatography and electron microscopy. Again, at 1 C the unfolding inducedby HHP proved to be reversible, with Gf = -2.4 kcal/mol and V = 72 mL/mol. Interestingly, at 25C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completelyblocks its aggregation under HHP. These data are relevant to the process by which the portal proteinassembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee theproper ring formation.